High‐Speed Large‐Field Multifocal Illumination Fluorescence Microscopy

Abstract

Scanning optical microscopy techniques are commonly restricted to a sub‐millimeter field‐of‐view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large‐field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam‐splitting grating and an acousto‐optic deflector synchronized with a high‐speed camera to attain real‐time fluorescence microscopy over a centimeter‐scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide‐field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide‐field microscopes to mitigate image blur due to tissue scattering and attain optimal trade‐off between spatial resolution and FOV. It further establishes a bridge between conventional wide‐field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large‐scale fluorescent‐based biodynamics.

Single-shot 3D wide-field fluorescence imaging with a Computational Miniature Mesoscope

“…S7, which demonstrates the mesoscopic imaging capability and the limitation of the current version of the CM 2 . The future generations of CM 2 will improve the light efficiency by exploring alternative designs, such as using novel focusing optics ( 12 ), diffractive optical elements ( 32 ), or fiber optics–coupled light sources ( 33 ). Second, the imaging optics of the CM 2 is designed by heuristically balancing several hardware and imaging attributes, including the resolution, FOV, image contrast, and device complexity and size.…”

Detection of cerebral tauopathy in P301L mice using high-resolution large-field multifocal illumination fluorescence microscopy

“…The LMI fluorescence imaging system is based on a beam-splitting grating and an acoustooptic deflector, which are synchronized with a high speed camera to attain real-time fluorescence microscopy over a 20×20 mm FOV [29,30]. A 473 nm CW laser was used for h-FTAA excitation.…”

“…The aim of the present study is to assess the selectivity and specificity of LCOs [28] for in vivo tauopathy detection in the P301L (Thy1.2) mouse model [16] with large-field multifocal illumination (LMI) fluorescence microscopy attaining 12×12 mm FOV and high spatial resolution (~6 m) [29,30].…”

Transcranialin vivodetection of amyloid-beta at single plaque resolution with large-field multifocal illumination fluorescence microscopy

“…Our recently developed LMI fluorescence imaging method based on a beam-splitting grating and an acousto-optic deflector synchronized with a high speed camera was employed for this study ( Fig. 1A) (35). Briefly, a high-repetition pulsed Q-switched, diode end-pumped Nd:YAG laser (model: IS8II-E, EdgeWave, Germany) operating at 532 nm wavelength was used for the excitation.…”

“…We devised a large-field multi-focal illumination (LMI) fluorescence microscopy method that provides a unique combination between an extended 20 × 20 mm field-of-view as well as high spatial (~20 m) and temporal (10 Hz) resolutions (34,35). In the present study, we demonstrated whole brain mapping of Aβ deposits at single plaque resolution in APP/PS1 (26) and arcA (36) mouse models of AD cerebral amyloidosis mediated by HS-169 LCOs (37).…”